Radio-methyl vorozole and methods for making and using the same

ABSTRACT

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

This application is a divisional of U.S. patent application Ser. No.12/704,114, filed Feb. 11, 2010, which claims priority from U.S.Provisional Application 61/152,356 filed on Feb. 13, 2009, both of whichare incorporated herein by reference in their entirety.

This invention was made with Government support under contract numberDE-AC02-98CH10886, awarded by the U.S. Department of Energy andsponsored by the National Institutes of Health under grant number K05 DA020001. The Government has certain rights in the invention.

FIELD OF INVENTION

Specific radiotracer vorozole compounds for in vivo and in vitroassaying, studying and imaging cytochrome P450 aromatase enzymes inhumans, animals, and tissues and methods for making and using the sameare provided.

BACKGROUND OF THE INVENTION

Cytochrome P450 aromatase, is the last enzyme in estrogen biosynthesis,catalyzing the conversion of androgens to estrogen. It plays a majorrole in the sexual differentiation of the brain during development andhas been implicated in the brain response to injury and in thepathophysiology of Alzheimer's disease. The enzyme is highly expressedin liver, steroidogenic organs and specific regions of the brainincluding the amygdala, in the bed nucleus of the stria terminalis, thepreoptic area (POA) and anterior hypothalamus. Moderate or lower levelshave been observed in many other brain regions including posterior andlateral hypothalamic nuclei, hippocampus and temporal cortex of rodents,non-human primates and humans.

Aromatase activity can be inhibited reversibly or irreversibly bysteroidal as well as non-steroidal compounds. Non-steroidal andsteroidal aromatase inhibitors are used as adjuvant therapies forpost-menopausal women having estrogen-dependent breast cancer. Examplesof non-steroidal aromatase inhibitors approved for such use includeletrozole (Femara®) and anastrozole (Arimidex®). These drugs have beenshown to reduce the rate of recurrence of cancer in treated patients.

Aromatase inhibitor drugs are also used by body builders and athleteswho abuse anabolic steroids, as a means of limiting the estrogenic sideeffects of excess androgens.

Radiotracer compounds that interact with the physiological targets ofthese drugs are useful for developing new forms of aromatase inhibitorsas well as for monitoring the treatment efficacy of the drugs and foradjusting dosages.

Radiotracer compounds that specifically interact with P450 aromataseenzymes are further useful for studies on the location of the enzyme intissue samples, for studying the enzyme mechanism of action and forkinetic studies on the synthesis and turnover of the enzyme in tissues.

Previous attempts to make use of [N-methyl-¹¹C]vorozole as a reliable,highly specific imaging tracer in positron emission tomography (PET)studies have been inexplicably unsuccessful, with the radiotracerexhibiting low regional specificity and high non-specific binding (seeLidström, et al. (1998) Nucl. Med. Biol. 25:497-501; Takahashi, et al.(2006) Neuroreport 19:431-435).

In addition, an attempt to make use of [¹¹C]letrozole as a specificradiotracer for studying brain aromatase enzymes in vivo (Kil, et al.(2008) J. Nucl. Med. 49 (Sup. 1):285P) found that the tracer was rapidlytaken up in the brain and was then rapidly cleared. Pretreatment withunlabeled letrozole failed to block uptake. The rapid clearance of the[¹¹C]letrozole and lack of specific binding indicated that it was not auseful radiotracer for brain aromatase activity.

Thus, previous attempts to use radiotracer-labeled aromatase inhibitorsto study aromatase activity in the brain and other tissues have beenrelatively, but inexplicably, unsuccessful.

SUMMARY OF THE INVENTION

(S)-Vorozole(6-[(S)-(4-chlorophenyl)-1H-1,2,4-triazol-1-ylmethyl]-1-methyl-1H-benzotriazole)(structure 1) is a specific and potent non-steroidal aromatase inhibitor(Wouters, et al: (1989) J. Steroid. Biochem. Mol. Biol. 32:781-788;Decoster, et al. (1990) J. Steroid. Biochem. Mol. Biol. 37:335-341)originally developed as an antineoplastic agent (Decoster, et al. (1992)Cancer Res. 52:1240-1244).

Standard synthetic routes to the preparation of vorozole racemicmixtures as well as methods for producing the enantiomeric (S)stereoisomer are well known. For example, see U.S. Pat. No. 4,943,574,the contents of which are incorporated herein by reference, DeKnaep, etal. (2000) Org. Process Res. & Devel. 4:162-166, and Venet, et al.(1997) Actualités de Chimie Therapeutique 23:239-246. These methodsgenerally make use of N-1 methyl-benzotriazole and its derivatives as astarting material. Thus the methyl group in vorozole is located in theN-1 position of the benzotriazole ring.

To develop a route to preparing vorozole as a ¹¹C positron emissiontomography (PET) radiotracer it was practical to use ¹¹C-methyl iodideto methylate a precursor compound lacking the N-1 benzotriazole methylgroup. Thus norvorozole (S)-norvorozole(5-[(S)-(4-chlorophenyl)-1H-1,2,4,-triazol-1-yl)methyl]-1H-benzotriazole)(structure 2) was used as the precursor over a decade ago to makecarbon-11 labeled vorozole via N-alkylation with [¹¹C]methyl iodide(Lidström, et al. (1998)) for positron emission tomography (PET) imagingand in vitro studies as a research tool for studies of aromatase inbrain and peripheral organs.

Lidström et al. (1998) reported that the alkylation reaction resulted inthe formation of two isomers of vorozole, [N-methyl-¹¹C]vorozole, (i.e.,the N-1 isomer, structure 3) and the N-3 isomer (structure 4) in a 5:2ratio. They reported that ¹¹C vorozole was well separated from the N-3isomer under the HPLC conditions employed.

Despite the purported purity of the ([N-methyl-¹¹C]vorozole) synthesizedby the Lidström et al. (1998) procedure, the radiotracer showed highliver uptake and “an otherwise relatively even distribution ofradioactivity”. They further found that blocking with unlabeled vorozolepretreatment caused only minor alterations of the biodistribution of theradiotracer. Takahashi, et al. (2006), using similar synthetic andapparently similar purification methods, found low target-to-backgroundratios (e.g. amygdala to cerebellum ratio of 1.25) and variability inblockable uptake in the preoptic area. However, the radiotracer showedremarkable metabolic stability in plasma and reasonable brainpenetration.

In the present work, a mixture of compounds was also found uponreproducing the original synthesis and purification conditions for C-11labeled vorozole. Furthermore, the labeled vorozole prepared asdescribed by Lidström et al. (1998) showed low regional specificity andhigh non-specific binding in baboon brain imaging studies.

The imaging results with the labeled compound did not correlate wellwith the known specificity of pure vorozole, which, as noted herein isroutinely synthesized using a precursor having the methyl group locatedat the N-1 position of the benzotriazole ring. The imaging results alsofailed to correlate with other methods that identified brain regionshigh in aromatase mRNA and aromatase activity (Abdelgadir, et al. (1997)Biol. Reprod. 57:772-777). Our laboratory's additional demonstrationthat [¹¹C]letrozole failed to provide a reliable radiotracer foraromatase activity, suggested that use of [¹¹C]-labeled aromataseinhibitors was less sensitive to levels of aromatase in vivo than theother, in vitro methods used to measure levels of aromatase. However,while not clarifying the reason for the poor results with[¹¹C]letrozole, an alternative explanation of our results and theearlier reports with [¹¹C]vorozole was that the fraction previouslythought to be pure [¹¹C]vorozole could be a mixture of compounds.

A detailed investigation of the alkylation of norvorozole (structure 2)with both [¹²C] and [¹¹C]methyl iodide, revealed that the originalsynthesis resulted in three isomers, not two isomers, and that the wellseparated labeled side product previously reported as the N-3 isomer wasactually the N-2 isomer (structure 5).

Moreover, under the reported HPLC conditions (Lidström, et al. 1998),the N-3 isomer (structure 4) co-eluted with [N-methyl-¹¹C]vorozole (theN-1 isomer, structure 3) and thus affected PET imaging and analysis.

The specific separation of pure [N-methyl-¹¹C]vorozole (N-1 isomer) fromthe heretofore unrecognized contaminating ¹¹C labeled N-3 isomerresulted in a reliable and highly specific in vivo radiotracer forcytochrome P450 aromatase enzymes.

Thus, one form of the present invention is directed to N-1 radio-methylvorozole (structure 6, wherein *C represents an isotope of carbon and^(§)H represents an isotope of hydrogen) substantially separated fromthe N-3 radio-methyl isomer (structure 7, wherein *C represents anisotope of carbon and ^(§)H represents an isotope of hydrogen).

The radio-methyl labeled vorozole, substantially separated from theradio-methyl N-3 isomer, may be labeled with an isotope of carbon (*C),including carbon-11 (¹¹C), carbon-13 (¹³C) and carbon-14 (¹⁴C).Alternatively the radio-methyl labeled vorozole may be labeled with anisotope of hydrogen (^(§)H), including deuterium (D) or tritium (³H).Further, the radio-methyl labeled vorozole may be dual labeled with anisotope of carbon as well as an isotope of hydrogen or with a mixture ofisotopes of carbon or with a mixture of isotopes of hydrogen andcombinations thereof.

Another embodiment of the present invention includes the methods forsubstantial separation of radio-methyl vorozole from the radio-methylN-3 isomer. The methods include analytical scale separation methods aswell as preparative scale separation methods. The methods furtherinclude rapid methods useful in preparation of the ¹¹C-labeled vorozolewhere time is of the essence as well as less rapid methods which may beuseful for preparing radio-methyl labeled vorozole with the long-livedisotopes (¹³C, ¹⁴C, D and ³H).

In addition this invention provides means for using the radio-methyllabeled vorozole, substantially separated from the radio-methyl N-3isomer. The means of using the compound include its use in positronemission tomography (PET)(¹¹C labeled), magnetic resonance imaging (MRI)(¹³C labeled), autoradiography in tissue sections (¹⁴C labeled, and/or³H labeled) and enzyme kinetics and mechanistic studies (D labeled).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A. Preparative HPLC profile of the crude reaction mixture fromthe alkylation of norvorozole with [¹¹C]methyl iodide. HPLC conditions:Spherisorb ODS (2) (Phenomenex, 5μ, 250 mm×10 mm); eluent, ammoniumformate buffer (50 mM, pH 3.5):acetonitrile (65:35), 5 mL/min. FractionX was isolated and subjected to analytical HPLC. B. Comparison of HPLCanalysis of the isolated “vorozole” fraction X, in the Lidström, et al.(1998) system (top graph: Spherisorb ODS (1), (Phenomenex, 5μ, 250×4.60mm); eluent, ammonium formate buffer (50 mM, pH 3.5):acetonitrile(40:60), 1 mL/min) and in the new HPLC system (lower graph: Luna PFP(2),(Phenomenex, 5μ, 250 mm×4.6 mm); eluent, aqueous formic acid (pH3.0):methanol (1:2), 1 mL/min).

FIG. 2: Preparative HPLC profile of the crude reaction mixture from thealkylation of norvorozole with [¹¹C]methyl iodide. Baseline separationof [N-methyl-¹¹C]vorozole from the N-2 and N-3 isomers was achieved.HPLC conditions: Luna PFP(2) (Phenomenex, 250 mm×10 mm, 5μ); aqueousformic acid adjusted to pH=3)/methanol (45/55); 5 ml/min.

FIG. 3: PET images in the (transaxial (top row) and coronal (bottom row)planes in baboon brain summed from 15-90 min after injection for (A) the15 minute fraction (X), (mixture of structure 3 and structure 4, 176.49MBq (4.77 mCi)), (B) [N-methyl-¹¹C]vorozole (structure 3, 163.91 MBq(4.43 mCi)); (C) N-3 isomer (structure 4, 123.21 MBq (3.33 mCi)) and (D)N-2 isomer (structure 5, 44.77 MBq (1.21 mCi)). All PET images aredose-corrected. Arrows indicate the amygdala (Amg) and preoptic area(POA).

DETAILED DESCRIPTION OF THE INVENTION

Reinvestigation of the synthesis and purification of[N-methyl-¹¹C]vorozole showed that all three benzotriazole isomers(vorozole, i.e., N-1 (structure 3); N-2 (structure 5); and N-3(structure 4)) are produced. Each of the isomers was characterized by¹³C-NMR and by 2D-NOESY spectra. A rapid HPLC system to substantiallyseparate [N-methyl-¹¹C]vorozole from the [¹¹C]labeled N-3 isomer(structure 3) using a pentafluorophenylpropyl bonded silica HPLC columnwas developed. Positron emission tomography (PET) studies in non-humanprimates of the pure [N-methyl-¹¹C]vorozole revealed accumulation ofC-11 in all brain regions with highest accumulation in the aromataserich amygdala and preoptic area (POA). Accumulation in these brainregions as well as other brain regions could be blocked by pretreatmentwith vorozole or letrozole, in accordance with reports indicating thatsome level of aromatase expression is present in many brain regions.

The significant image degradation caused by the contamination of thelabeled vorozole (structure 3) with an equal amount of the N-3 isomer(structure 4) brings to light the fact that [N-methyl-¹¹C]vorozole,substantially separated from the [¹¹C] N-3 isomer, will be asignificantly better radiotracer than earlier studies had indicated.These findings necessitate the re-evaluation [N-methyl-¹¹C]vorozole datafrom previous studies, both in vivo and in vitro. The availability ofpure [N-methyl-¹¹C]vorozole for PET represents a new scientific tool forstudies of the biology of aromatase and for drug research anddevelopment.

An initial research goal was to reproduce the previous radiosynthesisand HPLC conditions (observation of two radioactive fractions) and thento separate the “vorozole” fraction (X) if it was found that itcomprised a mixture of vorozole and another isomer as suggested in trialresults with non-radioactive methyl iodide. Since the HPLC column(Spherisorb ODS1, Beckman, 250 mm×10 mm) used previously was notcommercially available, it was replaced with a similar HPLC column(Spherisorb ODS (2), Phenomenex, 5μ, 250 mm×10 mm).

Using this HPLC column only two radioactive fractions in a ratio of 2:1were observed. The major radioactive fraction X (retention time 15minutes) (FIG. 1A), which has the same retention time as authenticvorozole, was isolated and subjected to further separation analysis(FIG. 1B) using analytical HPLC systems. A similar HPLC column(Spherisorb ODS (1), Phenomenex, 5μ, 250×4.60 mm) to that reportedpreviously (Lidström, et al. 1998) failed to reveal any heterogeneity inX as it showed only one peak in this system (FIG. 1B, upper graph). Manyother conventional ODS columns gave the same results using varioussolvent systems.

However, a pentafluorophenylpropyl (PFPP) bonded-phase HPLC columnresolved the 15 min fraction X into two peaks having the same retentiontimes as a preparation of unlabeled N-3 isomer (structure 4) and asvorozole (structure 1 and 3) (FIG. 1B, lower graph).

All three isomers of the crude reaction mixture were well-separatedusing a semi-preparative scale PFPP column (FIG. 2). The radiochemicalyields of the three isomers were approximately 30% each. Theradiochemical purity was >97% and the specific activity ranged 10-19Ci/μmol (0.3-0.7 GEq/μmol). The total radiosynthesis and separation timewas 65 min after the end of the cyclotron bombardment (EOB).

In one embodiment, radio-methyl vorozole, substantially separated fromthe radio-methyl N-3 isomer of vorzole is provided. The radio-methylvorozole, substantially separated from the radio-methyl N-3 isomer ofvorzole, may contain a radioisotope of carbon. The radioisotope ofcarbon may be ¹¹C, ¹³C or ¹⁴C. The radio-methyl vorozole, substantiallyseparated from the radio-methyl N-3 isomer of vorzole, may contain aradioisotope of hydrogen. The radioisotope of hydrogen may be deuterium(D) or tritium (³H). The radio-methyl vorozole, substantially separatedfrom the radio-methyl N-3 isomer of vorzole, may contain a mixture ofisotopes, including a mixture of isotopes of carbon, a mixture ofisotopes of hydrogen and/or a mixture of isotopes of hydrogen andisotopes of carbon.

Radio-methyl vorozole, substantially separated from the radio-methyl N-3isomer, is a preparation of radio-methyl vorozole that comprises greaterthan 50% mole fraction radio-methyl vorozole and less than 50% molefraction N-3 isomer. Preferably radio-methyl vorozole, substantiallyseparated from the radio-methyl N-3 isomer, is a preparation ofradio-methyl vorozole that comprises greater than 70% mole fractionradio-methyl vorozole and less than 30% mole fraction N-3 isomer. Morepreferably radio-methyl vorozole, substantially separated from theradio-methyl N-3 isomer, is a preparation of radio-methyl vorozole thatcomprises 90% or greater mole fraction radio-methyl vorozole and 10% orless mole fraction N-3 isomer.

Isomers of vorozole, i.e., vorozole, in which the methyl group is at theN-1 position of the benzotriazole ring; the N-2 isomer in which themethyl group is at the N-2 position of the benzotriazole ring; and theN-3 isomer in which the methyl group is at the N-3 position of thebenzotriazole ring may be designated variously as isomers, topoisomersand/or regioisomers and each designation is intended to convey the samemeaning.

Methods of preparing radio-methyl vorozole, substantially separated fromthe radio-methyl N-3 isomer, include analytical scale and preparativescale chromatographic procedures.

Substantial separation of vorozole from the N-3 isomer of vorozole makesuse of high performance liquid chromatography resins that providemultiple mechanisms for selectivity. The resins for substantialseparation of the isomers provide a combination of at least twomechanisms for selectivity including hydrogen bonding, dipole-dipoleinteraction, aromatic Π-Π interaction and hydrophobic interaction. Manysuch resins are well known in the art and generally may include,variously, aromatic groups, alkyl groups and halogen groups tofacilitate the combination of separation mechanisms. One example used inthe present study was the pentafluorophenylpropyl-bonded Luna® PFP,which afforded a rapid substantial separation of the vorozole from theN-3 isomer.

Another example used in the present study was phenoxypropyl bondedcolumn (Synergi™ PolarRP) which afforded a less rapid, but substantialseparation of vorozole from the N-3 isomer. In the pheonxypropyl columnseparation the retention times for both isomers was greater than 50minutes using aqueous formic acid (pH=3.5):methanol (50:50) as theeluent. This latter resin may be suitable for separation of radio-methylvorozole from the radio-methyl N-3 isomer when long lived radioisotopes(¹³C, ¹⁴C, ³H and D) are used.

While the systems demonstrated here to substantially separateradio-methyl vorozole from the radio-methyl N-3 isomer were reversephase high performance liquid chromatography systems, it is possiblethat normal phase high performance liquid chromatography systems may bedeveloped to provide suitable combinations of hydrogen bonding,dipole-dipole interaction, aromatic Π-Π interaction and hydrophobicinteraction to effect the separation.

An analytical scale procedure may include two chromatography steps. Themethod includes providing of a reaction mixture for methyl-labeling ofnorvorozole comprising norvorozole and radio-methyl iodide, whereinradio-methyl iodide (*C^(§)H₃I) contains either isotopes of carbon (*C)or isotopes of hydrogen (^(§)H) or mixtures thereof, a firstchromatography step to separate a mixture comprising the radio-methylvorozole and the radio-methyl N-3 isomer from the radio-methyl N-2isomer and a second chromatography step to substantially separateradio-methyl vorozole from the radio-methyl N-3 isomer.

In this two step analytical procedure, the first chromatography step maybe performed on a number of HPLC chromatographic supports using avariety of conditions. Conventional ODS columns may be usedinterchangeably to effect the separation of the N-1, N-3 mixture ofisomers from the N-2 isomer. A support of Spherisorb ODS with anammonium formate buffer (50 mM, pH 3.5):acetonitrile (65:35) eluentprovides a mixture of radio-methyl vorozole and the radio-methyl N-3isomer separated from the radio-methyl N-2 isomer.

The second chromatography step to separate radio-methyl vorozole fromthe radio-methyl N-3 isomer makes use resins having multiple mechanismsof selectivity and which provide a combination of mechanisms ofseparation. Such resins generally include combinations of one or more ofaromatic groups, halogen groups and alkyl groups. Apentafluorophenylpropyl (PFPP) bonded silica column may be used. A LunaPFP (2) support may be used with an aqueous formic acid (pH 3):methanol(1:2) eluent.

Alternatively, the second chromatography step to separate radio-methylvorozole from the radio-methyl N-3 isomer may make use of Synergi™ PolarRP with aqueous formic acid (pH=3.5):methanol (50:50) as the eluent.

Preferably, analytical or preparative scale separation methods mayinvolve a single high performance liquid chromatography procedure toseparate radio-methyl vorozole from the N-2 and the N-3 radio-methylisomers. The method includes provision of a reaction mixture formethyl-labeling of norvorozole comprising norvorozole and radio-methyliodide. Following the labeling reaction, the mixture is subjected tochromatography on an HPLC resin offering multiple mechanisms forselectivity and therefore, a combination of separation mechanisms. Suchresins generally are comprised of aromatic groups (e.g., phenyl) andhalogen groups (e.g., fluorine) and alkyl groups (e.g., propyl, butyl,hexyl, dodecyl, octadecyl, etc.). Examples of such resins includepentafluorophenylpropyl (PFPP) bonded silica resins, such as LunaPFP(2). The eluent is comprised of aqueous formic acid (pH 3):methanolmixtures in ratios from about 1:1 to about 1:2 and preferably about1:1.5.

Radio-methyl vorozole, substantially separated from the N-3 radio-methylisomer, may be used in a variety of studies. When the compound islabeled with the positron emitting ¹¹C atom, the compound is suitablefor imaging aromatase in living systems using positron emissiontomography (PET). Preparation of the [N-methyl ¹¹C] vorozole,substantially separated from the [¹¹C]-N-3 isomer, for use in suchstudies includes removal of the HPLC solvent, for example byevaporation, followed by resuspension of [N-methyl ¹¹C] vorozole,substantially separated from the N-3 isomer, in a suitable aqueoussolution, for example physiological saline and sterilization, forexample by filtration.

Animal subjects are prepared for PET imaging using well knownprocedures. Catheters may be inserted in a suitable artery and a veinfor arterial sampling and radiotracer injection respectively. DynamicPET imaging may be carried out using commercially available PET camerasand associated data collection systems, analyzing equipment andsoftware. Doses of [N-methyl ¹¹C] vorozole may range from about 1 toabout 5 mCi, with a specific activity ranging from about 10 to about 25Ci/μmol with an injected mass ranging from about 0.05 to about 0.5 nmol.Dynamic scanning is carried out for a suitable period of time, generally30 to 90 minutes. Arterial sampling may be performed to obtain the timeactivity curve in plasma and to analyze selected samples for thefraction of C-11 present as unchanged parent compound. During the PETscan, subjects are monitored for heart rate, respiration rate, PO₂ andtemperature.

The PET imaging may be whole body imaging of the subject animal.Alternatively the PET imaging may be limited to a specific portion ofthe body of the imaged subject. A specific portion of the body of theimaged subject may be the brain, and/or specific sub-structures thereof;various glands of the subject, including adrenal glands, pituitaryglands, ovaries and testes; the lymphatic system of the subject,including the lymph nodes; the pulmonary system, including the lungs andheart; various organs, including the liver and kidneys; and, where thesubject animal is a mammal, the breasts.

The results of the PET imaging studies may be used for assessment of theefficacy of reduction of aromatase activity in subjects, for example,breast cancer patients treated with aromatase inhibitors. Displacementor blockade of [N-methyl ¹¹C] vorozole from binding aromatase bypre-administration of varying dosages of known or new aromataseinhibitors can be used to assess the efficacy and dose requirements ofthe aromatase inhibitors. This is useful in the development of new drugsto inhibit aromatase in patients with estrogen-sensitive cancers.

In such procedures to determine the efficacy of a dose of an aromataseinhibitor, generally a target tissue is selected for imaging the bindingof the radiotracer [N—¹¹C-methyl]vorozole (substantially separated fromthe N-3 radio-methyl isomer) and a baseline PET scan is obtained afterinjection of the radiotracer. After the ¹¹C has been allowed to decayfor three to ten, or more, half-lives (e.g., one to six hours), achallenge dose of the aromatase inhibitor is administered at apre-determined period of time prior to a second injection of theradiotracer [N—¹¹C-methyl]vorozole (substantially separated from the N-3radio-methyl isomer) is administered and a second PET scan is obtained.The efficacy of the aromatase inhibitor and efficacy of the specificdose administered can be assessed by comparing the images from thebaseline scan (first scan) with the images in the challenge scan (secondscan) to determine how well or poorly the challenge dose diminishedbinding by the radiotracer.

Because aromatase enzymes are also located in the brain, particularly inthe amygdala and the preoptic area of non-human primates, [N-methyl ¹¹C]vorozole, substantially separated from the N-3 isomer, is useful for PETimaging assessment of the activity levels of aromatase in relationshipto estrogen effects on neuronal health. It is potentially useful as atracer for changes in aromatase in the brain that occur during (or as aresult of) the development of Alzheimer's disease. It is further usefulfor studies examining the role of estrogens in neuro-protectionfollowing brain injury.

When [N-methyl ¹³C] vorozole, substantially separated from the N-3isomer, is prepared it may be used in Magnetic Resonance Imaging (MRI)scans of subjects. Preparation for injection into a subject is asdescribed for the [N-methyl ¹¹C] vorozole, including resuspension in anaqueous medium and sterilization. Although such studies remain to beperformed it is likely that MRI of vorozole in animals will be usefulfor examination of brain responses to brain injury and in studyingneurodegenerative diseases, such as Parkinson's disease and Alzheimer's.

When [N-methyl ¹⁴C] vorozole or [N-methyl ³H] vorozole, substantiallyseparated from the radio-methyl N-3 isomer, are prepared they may beused for autoradiographic experiments on tissue sections using standardmethods well known in the art. The location of aromatase enzymes withinpreserved tissue samples can be determined using a combination ofautoradiography and microscopy.

In such studies, suitable tissue preparations include breast biopsies,brain biopsies and other tissue specimens. Such tissues may be flashfrozen and sectioned for slide preparation or may be preserved (e.g., informalin or other preservative solvent) and then sectioned for slidepreparation. The tissue sections are then incubated with [N-methyl ¹⁴C]vorozole or [N-methyl ³H] vorozole, substantially separated from theradio methyl N-3 isomer, under conditions suitable for binding of thearomatase enzymes in the tissue preparation. Following incubation, thetissue preparation is washed to remove excess labeled vorozole and animage is obtained using autoradiography, for example, using photographicfilm, phosphor imaging or a beta-imager. These results can then becorrelated to the gross anatomy derived from histological staining aswell as micro-structure of the tissue examined using microscopy.

When [N-methyl D] vorozole, substantially separated from the N-3 isomeris prepared, it may be used for in vitro examination of the mechanism ofthe inhibition of isolated aromatase enzymes by vorozole. It may furtherprove useful for studies examining the mechanism of interaction of otheraromatase inhibitors with the enzyme active site.

EXEMPLIFICATIONS General Methods

Vorozole and norvorozole, and their enantiomers were generously providedby Johnson & Johnson Pharmaceutical Research and Development (Beerse,Belgium). All other chemicals and solvents were purchased from AldrichChemical Company (Milwaukee, Wis., USA) and were used without furtherpurification.

NMR spectra were recorded using a Bruker Avance 400 MHz NMR spectrometer(Bruker Instruments Inc. Billerica, Mass., USA). GC-MS analyses wereperformed with an Agilent 6890/5973N GC/MSD system (AgilentTechnologies, Avondale, Pa., USA) equipped with a DB-5 column (30 mlength×0.250 mm ID, 5 μm film thickness; injector temperature; split;injection temperature, 280° C.; oven temperature, 280° C., isothermal;carrier gas, He; flowrate 1 mL/min). The temperature of source andquadrupole of mass spectrometer were 280° C., 180° C., respectively.

[¹¹C]Methyl iodide was produced using a PETtrace MeI Microlab (GEMedical Systems, Milwaukee, Wis., USA) from [¹¹C]carbon dioxide, whichwas generated from a nitrogen/oxygen (1000 ppm) target (¹⁴N (p,α)¹¹C)using EBCO TR 19 cyclotron (Advanced Cyclotron Systems INC. Richmond,Canada). High performance liquid chromatography (HPLC) purification wasperformed by a Knauer HPLC system (Sonntek Inc., Woodcliff Lake, N.J.,USA) with a model K-5000 pump, a Rheodyne 7125 injector, a model 87variable wavelength monitor, and NaI radioactivity detector.

During the radiosynthesis, C-11 was measured by three PIN diodedetectors (3×3 mm Si diode, Carroll Ramsey Associates, Berkeley, Calif.)equipped with a triple-channel amplifier (model 101-HDC-3; CarrollRamsey Associates, Berkeley, Calif.) and NI USB-6008 (NationalInstruments, Austin, Tex.). Radioactivity of [N-methyl-¹¹C]vorozole wasmeasured by a Capintec CRC-712MV radioisotope calibrator (Capintec Inc.,Ramsey, N.J., USA). ¹¹C Radioactivity of preparative HPLC samples wasmeasured by a Packard MINAXI γ 5000 automated gamma counter (PackardInstrument, Meriden, Conn.). All radioactivity measurements weredecay-corrected. Specific activity was measured by radioactivity/mass(Ci/μmol). For quality control, radiochemical purities were measured byanalytical HPLC using aqueous formic acid solution (0.1% (v/v),pH=2.8):methanol (1:2) at a flow rate 1 mL/min on a Luna PFP(2) (250mm×4.6 mm, 5μ; Phenomenex, Torrance, Calif.). For baboon PET studies, aformulation of [N-methyl-¹¹C]vorozole (and the N-1/N-3 mixture and themethyl [¹¹C] N-3 or N-2 isomers) in 4 mL of saline was used.

Analysis of reaction products from the reaction of norvorozole withunlabeled methyl iodide: Potassium carbonate (60 mg, 434 μmol) andmethyl iodide (180 mg, 0.8 mmol) were added to a solution of(S)-norvorozole(6-[(S)-(4-chlorophenyl)-1H-1,2,4-triazol-1-ylmethyl]-1H-Benzotriazole(structure 2), 1.3 mg, 4 μmol) in anhydrous acetonitrile (1 mL). Thereaction mixture was stirred at room temperature for 7 min. Afterevaporation of acetonitrile and excess of methyl iodide under reducedpressure, the crude product was extracted with ethyl acetate and driedwith anhydrous sodium sulfate, filtered, and then evaporated to drynessusing a rotary evaporator under reduced pressure. This crude mixture wasseparated by HPLC using aqueous formic acid solution (pH=3.0):methanol(45:55) at a flow rate 1 mL/min on a Luna PFP(2) (250 mm×4.6 mm, 5μ;Phenomenex, Torrance, Calif.). The three major fractions of UV activepeaks (N-3 isomer (structure 4, without ¹¹C), T_(R)=21 min; vorozole(structure 1) T_(R)=24 min; N-2 isomer (structure 5, without ¹¹C),T_(R)=35 min) were collected separately (ratio, 1:1:1), and evaporatedunder reduced pressure. The residual water was extracted with ethylacetate, dried with anhydrous sodium sulfate, filtered, and evaporatedto give three analytical samples. These samples were examined by ¹H and¹³C NMR.

Reaction of norvorozole with [¹¹C]methyl iodide: To (S)-norvorozole(structure 2, 1 mg, 3.22 pmol) in DMSO (300 μL) 5 M KOH (1 μL, 1.6 eq)was added. After vortexing for 30 sec, the reaction mixture wastransferred into a 1.5 mL V-shape reaction vessel. [¹¹C]methyl iodidewas transferred in a helium stream from the PETtrace MeI Microlab methyliodide system into this solution at room temperature and peak trappingwas observed by a pin-diode detector. After the vessel was heated to 90°C. for 3 min, the reaction mixture was cooled and diluted with HPLCeluent (1 mL). Preparative HPLC was performed using a method simulatingthe original publication method for the synthesis of[N-methyl-¹¹C]vorozole (Method A) and another to substantially separate[N-methyl-¹¹C]vorozole from the N-3 and N-2 isomers (Method B).

Method A (Partial Separation):

We slightly modified the original HPLC method of Lidström, et al. (1998)based on column availability. Briefly, the reaction mixture diluted withHPLC solvent (1 mL) was subjected to HPLC chromatography on a SpherisorbODS2 (Phenomenex, 250×10 mm, 5μ) column using aqueous ammonium formatesolution (50 mM, pH=3.5):acetonitrile (65:35), and a flow rate of 5mL/min. The major C-11 labeled fraction (fraction X, FIG. 1A) wascollected at the expected retention time (T_(R)=15 min) for vorozole andthe other minor radioactive fraction was collected at 22 min. The 15 and22 minute fractions were present in a ratio of about 2 to 1,respectively. The solvent was removed from the 15 minute fraction and itwas rechromatographed on a pentafluorophenylpropyl (PFPP) based column(Luna PFP(2), Phenomenex, 250×0.46 mm, 5μ) column with aqueous formicacid (0.1% (v/v, pH=2.8):methanol (1:2) and flow rate of 1 mL/min toreveal two peaks of equal radioactivity at 10 and 11 min (FIG. 1B).

Method B (One-Step, Complete Separation):

The crude product in DMSO was diluted with HPLC solvent (1 mL) andchromatographed using a solvent mixture of aqueous formic acid solution(0.1% (v/v), pH=2.8):methanol (45:55) at a flow rate 5 mL/min on a LunaPFP(2) column (Phenomenex, 250 mm×10 mm, 5μ). [N-methyl-¹¹C]vorozoleeluted at 24.5 min and was collected.

The HPLC solvent was removed by azeotropic evaporation with acetonitrileusing a rotary evaporator under the reduced pressure. The residue wastaken up by saline (4 mL), filtered through a 0.2 μm HT Tuffryn®membrane filter (Acrodisc® 25 mm Syringe Filter, Pall Life Sciences, AnnArbor, Mich.) into a sterile vial ready for the baboon study. Theradiochemical purity for [N-methyl-¹¹C] vorozole was >99% as determinedby TLC (solvent, acetonitrile:water:NH₄OH (conc.) (90:9:1) onMacherey-Nagel plastic back silica plates (R_(f): 0.6).

In separate syntheses, fractions containing the N-3 and the N-2 isomers(which eluted at 21.2 and 39.5 min, respectively) were collected.

Measurement of log D: Log D_(7.4) for the three ¹¹C isomers was measuredusing a published method (Ding et al. (2005) J. Neurochem. 94:337-351;Reichel, et al. (1998) Pharm. Res. 15:1270-1274). Briefly, an aliquot(50 μL) of the ¹¹C vorozole or isomer solution was introduced into amixture of 1-octanol (2.5 mL) and phosphate buffered saline (PBS, pH7.4; 2.5 mL). The mixture was vortexed for 2 min and then centrifuged at7000 rpm for 2 min. An aliquot (0.1 mL) of the clear octanol layer and1.0 mL of the buffer layer were sampled separately into two empty vialsand the amount of radioactivity was determined. Two mL of the octanollayer was transferred into a test tube containing 0.5 mL of freshoctanol and 2.5 mL of buffer. After vortexing and centrifuging in thesame way as the first measurement, radioactivity of the second batch wasmeasured. In general, these processes were repeated up to 6 times. LogD_(7.4) at pH=7.4. is as the log₁₀ of the average of the ratios of thedecay corrected counts in the octanol:phosphate buffer (pH=7.4).

Measurement of free fraction in plasma: An aliquot of each of the¹¹C-labeled isomers was incubated at room temperature for 10 min withbaboon plasma (500 μL) as described previously (Ding, et al. (2005)).Briefly, aliquots (20-40 μL) of the incubated spiked plasma were counted(unspun aliquots). A portion of the incubation mixture (200-400 μL) wascentrifuged using a Centrifree tube (Amicon Inc, Beverly, Mass., USA,molecular weight cut-off, 30,000) for 10 minutes. The top portion of theCentrifree tube (the bound portion) was removed and precisely measuredaliquots (20-40 μL) of the liquid in the cup (unbound fraction) werecounted. The free fraction is the ratio of radioactivity of the unboundaliquots to the radioactivity of the unspun aliquots. All counts weredecay-corrected.

PET Studies in Baboons: All animal studies were reviewed and approved bythe Brookhaven Institutional Animal Care and Use Committee. Fourdifferent baboons were studied in 7 PET sessions where two or threeradiotracer injections were administered two hours apart to comparedifferent isomers or to assess reproducibility of repeated measures orthe effects of a blocking dose of vorozole or letrozole (0.1 mg/kg, 5min prior). Baboons were anesthetized with ketamine (10 mg/kg) and thenintubated and ventilated with a mixture of isoflurane (Forane, 1-4%) andnitrous oxide (1500 mL/min) and oxygen (800 mL/min). Animals weretransported to and from the PET facility in a temperature controlledtransfer cage and a member of the staff attended them while theyrecovered from anesthesia. Catheters were inserted in a popliteal arteryand radial arm vein for arterial sampling a radiotracer injectionrespectively. Dynamic PET imaging was carried out on a Siemen's HR+ highresolution, whole body PET scanner (4.5×4.5×4.8 mm at center of field ofview) in 3D acquisition mode, 63 planes. A transmission scan wasobtained with a ⁶⁸Ge rotating rod source before the emission scan tocorrect for attenuation before each radiotracer injection. The injecteddose of ¹¹C compounds ranged from 55.5 to 185 MBq, the specific activityranged from 16 to 19 Ci/μmol at the end of the bombardment and theinjected mass ranged from 0.08-0.3 nmol. Dynamic scanning was carriedout for 90 minutes with the following time frames (1×10 sec; 12×5 sec;1×20 sec; 1×30 sec; 8×60 sec; 4×300 sec; 8×450 sec). Arterial samplingwas performed to obtain the time activity curve in plasma and to analyzeselected samples for the fraction of C-11 present as unchanged parentcompounds with the sample times described previously (Ding, et al.(2005)). During the PET scan, baboons were monitored for heart rate,respiration rate, PO₂ and temperature. Animals were allowed 4 weeksbetween studies to recover from anesthesia and blood sampling.

Plasma analysis for fraction of ¹¹C-isomers: Plasma samples at 1, 5, 10,30 and 60 min were analyzed by HPLC to determine the percent of residualparent tracer. Plasma samples from selected time points were added to300 μL acetonitrile containing an appropriate amount of standard, thensubjected to cell disruption using a Polytron (Brinkmann Instruments),centrifuged for 4 minutes and decanted into 300 μL water. HPLCconditions were 60:40 0.1 M ammonium formate:acetonitrile, WatersμBondapak column (3.9×300 mm), flow 1.3 mL/min, UV detection at 254 nm.Supernatants were injected onto the column, reserving a measured amountto determine recovery of injected activity from the column. Retentiontimes were 7.5 minutes for vorozole itself and the N-3 isomer, and 9.5minutes for the N-2 isomer. Five fractions were collected from eachinjection, 3 before the parent peak, the parent peak, and finally thetail of the parent peak. The percent unchanged tracer was determined bydividing the sum of the last 2 peaks by the sum of all HPLC fractions.Sample recoveries were determined to verify that there were no labeledmetabolites retained on the column.

Image Analysis: Time frames were summed over the 90 minute experimentalperiod. Regions of interest were placed over the amygdala, the preopticarea, frontal cortex, cerebellum and thalamus on the summed image andthen projected onto the dynamic images to obtain time activity curves.Regions occurring bilaterally were averaged. Carbon-11 concentration ineach region of interest was divided by the injected dose to obtain the %dose/cc. The summed PET images from one baboon was co-registered with a3D MR image of the same animal using PMOD (PMOD Technologies, Ltd.).

Results and Analysis:

Reaction of norvorozole (structure 2) with [¹²C]methyl iodide: Because[N-methyl-¹¹C]Vorozole synthesized by the published method (Lidström, etal, 1998) using slightly modified HPLC purification conditions, did notgive the high signal to noise baboon brain images and becauseautoradiography results with rat brain sections also showed non-specificbinding, we examined the products of the Lidström et al. (1998) reactionand purification methods more thoroughly using unlabeled methyl iodide.

Given the alkaline reaction medium, we first considered whether thenon-specific binding could be a result of racemization, resulting in theformation of the inactive (R)-vorozole (Wouters, et al. (1990) J.Steroid. Biochem. Mol. Biol. 37:1049-1054), which would have co-elutedwith the active form. However, chiral HPLC analysis (column, ChiralpakOJ-P, 4.6×15 mm; eluent, water:acetonitrile (75:35); flowrate, 0.5ml/min) indicated that no racemization of either (S)-norvorozole or(S)-vorozole occurred under the reaction conditions.

Serendipitously, during this investigation, we uncovered the presence ofan unexpected reaction product, was formed in approximately equal yieldto (S)-vorozole. This product was not separable under the HPLCconditions originally reported. Thus Lidström et al. (1998) noted onlytwo major fractions “in a ratio of 5 to 2”. The authors identified thetwo fractions as ([N-methyl-¹¹C]vorozole (N-1 alkylation) and an isomer,(N-3 alkylation isomer). Their assignment was based on ¹³C NMRexperiments, in which [¹³C]methyl iodide was added as a carrier in thecarbon-11 methylation reaction in order to have sufficient mass for a¹³C NMR. After HPLC isolation of the fraction with the expectedretention time of vorozole, the authors determined the chemical shift ofthe ¹³C-enriched product matched that of vorozole (34.4 ppm). TheLidström, et al. (1998) minor isomer, which was well separated undertheir HPLC conditions, was also isolated and analyzed by ¹³C NMR andassigned as the N-3 methylation product with a reported chemical shiftof 43.4 ppm.

When we reproduced the original radiosynthesis as closely as possible(Spherisorb ODS2 columns (Phenomenex) were used instead of SpherisorbODS1 (Beckman) because of commercial availability), we also found twoapparent C-11 labeled fractions in a similar ratio (2:1) (FIG. 1A).

Since there was precedent for alkylation at the N-2 position ofbenzotriazole rings (Kopanska et al. (2004) Bioorgan. Med. Chem.12:2617-2624), we investigated the possibility that the N-2 isomer mayhave formed and that it co-eluted with the labeled vorozole, thusaccounting for the low specificity and reproducibility we had observedin our PET studies. In fact, GC-MS analysis confirmed that threeproducts were formed by the reaction of norvorozole with excess methyliodide. Consistent with our hypothesis, each of the three isomers hadthe same molecular weight (M⁺=324) yet their fragmentation patternsdiffered distinctly. Later, we observed three singlet peaks between4.2-4.6 ppm in the ¹H-NMR spectrum. The ¹³C-NMR spectrum of the crudereaction mixture also confirmed three methyl peaks, 34.61, 34.66, and43.64 ppm. Thus, it was apparent that the reaction mixture containedthree different methylated compounds.

In spite of rigorous HPLC analysis with six different reversed-phase andtwo different normal-phase columns using several gradient elutionsystems, only two major fractions were observed. This prompted us toisolate and characterize the two major UV-active fractions from HPLCseparation in order to confirm that the vorozole fraction wascontaminated by one of the other isomers.

GC-MS and ¹³C-NMR analysis of the fraction assigned as vorozoleconfirmed the presence of two isomers of the same molecular weight with¹³C-methyl shifts of 34.61 and 34.66 ppm (125 MHz, CDCl₃), one of which(34.61 ppm) corresponded to the chemical shift of an authentic sample ofvorozole. These two shifts are so similar that they were probably notresolved at the lower field strength used in the original analysis(Lidström, et al. 1998). Thus the Lidström et al. (1998) rigorousanalysis, which has been typical in the field of carbon-11 labelingradiosynthesis was, in this instance, unable to distinguish the twoisomers and thus led to PET evaluation of a mixture, with concomitantlypoor results.

Complete resolution of the reaction mixture: To achieve theexceptionally difficult separation of the three isomers for fullcharacterization, we screened a number of methods for purification. Aninitial separation of the two co-eluting isomers from the ODS column wasachieved (T_(R)=52 and 58 min) using aqueous formic acid(pH=3.5)/methanol (50:50) at a flow rate (1 mL/min) on a silica basedphenoxypropyl bonded column (Synergi™ PolarRP, 5μ, 250×4.6 mm,Phenomenex). After isolation, each compound was characterized by ¹³C-NMRand 2D-NOESY NMR.

The byproduct that could be isolated in pure form using the original ODSHPLC conditions (¹³C of N-methyl, 43.64 ppm), and which was previouslyassigned as the N-3 methylated isomer (structure 4), was re-assigned asthe N-2 isomer (structure 5). This reassignment was primarily based onthe ¹³C chemical shift of the two quaternary carbons (both C-8 and C-9,above 140 ppm) of the benzoquinoid ring, which were shifted clearlydownfield relative to the corresponding one of benzotriazole carbonswhich is typically below 140 ppm (Carta, et al. (2005) Heterocycles65:2471-2481).

The other two isomers, which co-eluted under the original HPLCconditions but which were separated by the Synergi™ PolarRP column andthen by the PFPP column, were identified as vorozole (N-1 isomer,structure 3) and the N-3 isomer (structure 4). Their structuralassignments were largely based on the NOE (nuclear Overhauser effect)correlation of the H-7 or H-4 to the methyl group.

Comparison of C-11 labeled [N-methyl-¹¹C]vorozole and the ¹¹C-labeledN-2 and N-3 isomers and a mixture of [N-methyl-¹¹C]vorozole and¹¹C-labeld N-3 isomer in the baboon brain: All three regioisomers have alipophilicity (log D_(7.4)) considered suitable for blood-brain barrierpenetration. Notably, the log D's of vorozole and the N-3 isomer werealmost identical, which is consistent with their co-elution on HPLCusing the ODS column. The free fraction of the two isomers in baboonplasma was also similar.

FIG. 3 compares PET images of the 15 minute fraction (mixture of[N-methyl-¹¹C] vorozole and the labeled N-3 isomer), [N-methyl-¹¹C]vorozole, labeled N-3 isomer and labeled N-2 isomer at the levels of theamygdala (top row) and preoptic area (POA) (bottom row). These imagesshowed (1) low signal to background of the aromatase-rich amygdala andno specific uptake in the POA for the mixture; (2) high retention inamygdala and preoptic area for [N-methyl-¹¹C]vorozole and (3) nospecific uptake in amygdala and preoptic area for either of the isolatedN-3 or N-2 isomers. Low signal to background for the mixture isconsistent with contamination of the N-1 isomer whereas the high signalto noise in the amygdala and preoptic areas for pure vorozole (N-1isomer) was consistent with the high affinity of vorozole for aromatase.We note that affinity values for the two non-vorozole isomers foraromatase are not available though we would predict low affinitiesrelative to vorozole (IC₅₀=2.7 nM)[29] based on our imaging results (seeWouters, et al. (1989) J. Steroid. Biochem. Mol. Biol. 34:427-430).

Time-activity curves for pure [N-methyl-¹¹C]vorozole in the baboon brainrevealed very high initial uptake in five brain regions (amygdala,cerebellum, frontal cortex, white matter and preoptic area). Carbon-11peaked at ˜5 min and cleared to a low point at 20-30 min and relativelystable accumulation through the end of the study in all brain regions.Regions with the highest accumulation of radiotracer were the amygdalaand the preoptic area, both of which are rich in aromatase. Pretreatmentwith “cold” vorozole or letrozole reduced [N-methyl-¹¹C]vorozoleaccumulation in all brain regions examined, indicating its specificityto brain aromatase. After vorozole treatment there is a rapid but lowerinitial uptake followed by a steady clearance and no accumulation of[N-methyl-¹¹C]vorozole for both the amygdale and the preoptic brainregions. A similar pattern after pretreatment was seen for other brainregions.

Both of the other labeled isomers (N-3 and N-2) show reasonably highbrain penetration (0.02-0.03% ID/cc at 2.5 min, data not shown) but nospecific distribution to aromatase-rich brain regions.

Analysis of chemical form of C-11 in plasma after administration of[N-methyl-¹¹C]vorozole, labeled N-3 isomer and labeled N-2 isomer in thebaboon: For all baboon studies, total C-11 in plasma was measured andthe unchanged fraction of C-11 labeled compound was determined by HPLC.The % unchanged C-11 labeled vorozole in baboon plasma was high (up to78-80% at 90 min).

Interestingly, the position of the methyl group seems to exert aconsiderable effect on the metabolism, in that N-2 and N-3 isomersshowed very rapid appearance of labeled metabolites and inter-subjectvariability seemed high compared with labeled vorozole. However, thearea under curves (AUCs) for labeled vorozole and the labeled N-3 isomerin plasma normalized to the injected dose over 90 minutes were similar,suggesting that the contribution of the N-3 isomer in the PET image ofthe mixture would be significant. The labeled N-2 isomer showed a rapidrate of disappearance of the parent labeled compound from plasma.

Recent use of ¹¹CH₃ vorozole, substantially separated from the N-3radio-methyl isomer, in PET imaging of brains of healthy male and femalehuman volunteers showed a strikingly different distribution pattern thanthe pattern found in rodents and non-human primates. In humans thehighest levels were found in distinct thalamic nuclei and moderatelyhigh levels were found in amygdale, preoptic area and the medulla.

The invention claimed is:
 1. A method of preparing N-1 radio-methylvorozole substantially separated from an N-3 radio-methyl isomercomprising: a) providing a mixture comprising radio-methyl vorozole andradio-methyl N-3 isomer; b) applying said mixture, under suitableconditions, to a chromatography resin offering multiple mechanisms forselectivity; c) eluting said compounds using an eluent comprisingaqueous formic acid:methanol; thereby obtaining a composition consistingof N-1 radio-methyl vorozole.
 2. The method according to claim 1 whereinthe mixture optionally further comprises a radio-methyl N-2 isomer. 3.The method according to claim 1 wherein the multiple mechanisms ofselectivity include at least two of hydrogen bonding, dipole-dipoleinteraction, aromatic Π-Π interaction and hydrophobic interaction. 4.The method according to claim 3 wherein the resin comprises aromaticcomponents and alkyl components.
 5. The method according to claim 4wherein the resin optionally further comprises halogen components. 6.The method according to claim 5 wherein the halogen component isfluorine.
 7. The method according to claim 6 wherein the resin is apentafluorophenylpropyl (PFPP) resin.
 8. The method according to claim 4wherein the resin comprises a phenoxypropyl resin.
 9. The methodaccording to claim 1 wherein the eluent comprises aqueous formicacid:methanol in ratios varying between 1:1 and 1:2.
 10. The methodaccording to claim 9 wherein the ratio varies between 1:1 and 1:1.5. 11.The method according to claim 10 wherein the ratio varies between 1:1and 1:1.2.
 12. The method according to claim 1 wherein the aqueousformic acid is adjusted to a pH between about 2.0 and about 4.5.
 13. Themethod according to claim 12 wherein the pH is between about 2.5 andabout 3.5.
 14. The method according to claim 1 wherein the N-1radiomethyl vorozole and the N-3 radiomethyl vorozole isomer are[¹¹C]-radiomethyl radiomethyl vorozole and vorozole isomers,respectively.
 15. The method according to claim 14 wherein the¹¹C-vorozole and vorozle isomer are formed through methylation ofnorvorozole with [¹¹C]Methyl iodide.
 16. The method according to claim 2wherein the N-1 radiomethyl vorozole and the N-2 and the N-3 radiomethylvorozole isomers are [¹¹C]-methyl vorozole and [¹¹C]-methyl vorozoleisomers, respectively.
 17. The method according to claim 16 wherein the[¹¹C]-methyl vorozole and [¹¹C]-methyl vorozole isomers are formedthrough methylation of norvorozole with [¹¹C]Methyl iodide underconditions for complete methylation of norvorozole.
 18. The[¹¹C]N1-methyl vorozole prepared by any one of the methods of claim14-17.
 19. A method of preparing N1-radio-methyl vorozole substantiallyseparated from N2- and N3-radio-methyl isomers of vorozole comprising:a) providing a mixture comprising N1-radio-methyl vorozole,N2-radio-methyl isomer and N3-radio-methyl isomer; b) applying saidmixture, under suitable conditions, to a chromatography resin selectedfrom the group consisting of pentafluorophenylproply (PFPP) resin andphenoxypropyl resin; and c) eluting said compounds using an eluentcomprising aqueous formic acid:methanol; thereby obtaining a compositionconsisting of N1-radio-methyl vorozole.
 20. The method according toclaim 19 wherein the mixture of N1-radio-methyl vorozole,N2-radio-methyl isomer and N3-radio-methyl isomer is provided throughmethylation of norvorozole with radio-Methyl iodide.
 21. TheN1-radio-methyl vorozole prepared by the method of claim 20.